Non-pathogenic F18 E. coli  strain and use thereof

ABSTRACT

There is provided an isolated  E. coli  strain deposited at the International Depositary Authority of Canada (IDAC) on Jun. 20, 2013 and attributed accession number 200613-01. There is also provided methods of using this strain for preventing edema disease or diarrhea caused by an F18 pathogenic  E. coli  infection in an animal.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/414,964, filed Jan. 15, 2015, which is the National Stage ofInternational Application No. PCT/CA2013/000660, filed Jul. 18, 2013,which claims the benefit of U.S. Provisional Application No. 61/674,179,filed Jul. 20, 2012, the contents of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

This application relates to a non-pathogenic F18 E. coli strain and tothe use thereof.

BACKGROUND

Edema disease and diarrhea typically are common diseases amongst animalsraised by breeders. For example, edema disease in weaned piglets istypically caused by shiga toxin—encoding Escherichia coli (STEC) strainsencoding the Shiga toxin type 2e (Stx2e) (MacLeod et al., 1991, VetPathol 28:66-73), while secretory diarrhea in newborn and weaned pigletsis typically caused by enterotoxigenic Escherichia coli strains (ETEC)encoding for heat stable (STa, STb, EAST1) and/or heat labile (LT)enterotoxins (Gyles and Fairbrother, 2010, Escherichia coli. In:Pathogenesis of bacterial infections in animals, ed. Gyles C L, PrescottJ F, Songer G, Thoen C O, 4th ed., pp 267-308. Blackwell Publishing,Ames, Iowa; Nagy et al., 1997, Microb Pathog 22:1-11). Some pathogenicstrains express both the Stx2e genes and enterotoxin genes and thereforemay be capable of causing symptoms of edema disease and those ofdiarrhea in the same animal (STEC/ETEC) (Barth et al., 2007, Berl MunchTierarztl Wochenschr 120:307-316).

A deficiency associated with many conventional therapeutic orprophylactic compositions and methods for intestinal bacterialinfections associated with symptoms of edema disease and/or diarrheadisease is their low reliability or efficacy. There is therefore a needfor an improved therapeutic or prophylactic compositions and methods foredema disease and/or diarrhea disease.

SUMMARY

In one non-limiting broad aspect there is provided an isolatedEscherichia coli strain deposited at the International DepositaryAuthority of Canada (IDAC) on Jun. 20, 2013 and attributed accessionnumber 200613-01.

In another non-limiting broad aspect there is provided a method forpreventing a pathogenic F18 Escherichia coli (E. coli) intestinalinfection in an animal, comprising intestinal delivery to the animal ofan effective amount of a live E. coli strain deposited at theInternational Depositary Authority of Canada (IDAC) on Jun. 20, 2013 andattributed accession number 200613-01.

In another non-limiting broad aspect there is provided a method forpreventing edema disease or diarrhea caused by a pathogenic F18Escherichia coli (E. coli) infection in an animal, comprising intestinaldelivery to the animal of an effective amount of a live E. coli straindeposited at the International Depositary Authority of Canada (IDAC) onJun. 20, 2013 and attributed accession number 200613-01.

In one non-limiting embodiment, the live E. coli strain is in alyophilized form. In another non-limiting embodiment, the live E. colistrain is in association with a feed acceptable carrier, for example thelive E. coli strain may be diluted, incorporated into, or suspended inthe feed acceptable carrier. In another non-limiting embodiment, thelive E. coli strain is in frozen form.

These and other aspects and features of the present invention will nowbecome apparent to those of ordinary skill in the art upon review of thefollowing description of specific embodiments of the invention inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A detailed description of the embodiments of the present invention isprovided herein below with reference to the accompanying drawings inwhich:

FIG. 1: Is a non-limiting graphic that illustrates fecal excretion ofthe non-pathogenic F18 strain after administration of the livenon-pathogenic F18 strain according to variable dosages, shown as theinverse log of the average of logarithmic colony forming units (CFU).

FIG. 2: Is a non-limiting graphic that illustrates fecal excretion of anF18-STEC pathogenic challenge strain: comparison between animals havingbeen treated with the combined composition and control animals.

FIG. 3: Is a non-limiting graphic that illustrates colonization by theF18-STEC pathogenic challenge strain in different intestinal segments:comparison between animals having been treated with the combinedcomposition and control animals.

FIG. 4: Is a non-limiting graphic that illustrates immunologicaldetection of IgM against F18 fimbriae in serological samples: comparisonbetween animals having been treated with the combined composition andcontrol animals.

DETAILED DESCRIPTION OF EMBODIMENTS

Generally, porcine STEC and many porcine ETEC strains express the F18determinant. The F18 determinant is a fimbriae that facilitatesbacterial colonization of the mucosal surface of the intestine(Fairbrother et al. 2006, Postweaning Escherichia coli diarrhea andedema disease. In: Diseases of swine, ed. Straw B, Zimmermann J J,D'Allaire S, Taylor D J, 9th ed., pp. 649-662. Blackwell Publishing,Ames, Iowa). It is believed that F18 fimbriae mediate adhesion of an F18expressing bacterial cell to an enterocyte receptor, the ECF18Rreceptor, which is exposed by the enterocytes of the small intestine ontheir apical cytoplasmic membrane (Vögeli et al. 1996, Anim Genet27:321-328; Waddell et al. 1996, Infect Immun 64:1714-1719).

Some porcine STEC and STEC/ETEC strains can express other cytoadhesivefimbriae or nonfimbrial adhesins in addition to the F18 fimbria, inparticular F4 or F5 fimbriae or the “adhesin involved in diffuseadherence” (AIDA) (Barth et al., 2007, Berl Munch Tierarztl Wochenschr120:307-316; DebRoy et al. 2009, J Vet Diagn Invest 21:359-364;Fairbrother et al., supra; Niewerth et al.: 2001, Clin Diagn Lab Immunol8:143-149). While the AIDA adhesin occurs frequently in both pathogens,F4 or F5 fimbriae are rare in those strains.

On farm, post-weaning diarrhea (PWD) and edema disease (ED) in swinegenerally occur in the first 2 weeks post-weaning and are associatedwith infection by pathogenic F4 or F18 enterotoxigenic E. coli (ETEC)strains for PWD and by pathogenic F18 Shiga toxin producing E. coli(STEC) strains for ED. For both strains, the fimbriae (i.e., F4 or F18)allow adhesion of the bacteria to their specific receptors, located onsmall intestinal villi, followed by division of the bacteria andcolonization of the intestine.

In PWD, F4 receptors are expressed in about 30 to 40% of pigs and F18receptors are detected in about 70 to 80% of pigs. The pathogen strainsproduce a combination of several toxins such as LT, STa and STb. Pigsinfected with ETEC-F4 generally show very high morbidity, delay ingrowth, watery and projectile diarrhea which often lead to the infectedanimal's death.

For ED, the F18 receptors are detected in about 70 to 80% of pigs. Thetoxin associated to STEC is Stx2e, a vasotoxin that acts on vascularendothelial cells resulting in edema and subsequent neurological signsincluding ataxia, decumbency and eventually, as for PWD, often lead tothe infected animal's death.

As used herein, the expression “F18 E. coli” or “F18 strain” relates toan E. coli strain that expresses the F18 determinant (fimbriae).

As used herein, the expression “F4 E. coli” or “F4 strain” relates to anE. coli strain that expresses the F4 determinant (fimbriae).

The person of skill will recognize that the herein describednon-pathogenic F18 strain may be manipulated so as to obtain a mutant orvariant strain thereof, a strain that has all the identifyingcharacteristics of the strain described herein, without undue effort.Methods of creating mutants or variants are common and well known in theart. For example, U.S. Pat. No. 7,371,558 (which is herein incorporatedby reference in its entirety) discloses a summary of some methods forcreating a “mutant or variant thereof.” Specific methods for creatingmutants using radiation or chemical agents are also well documented inthe art. See, for example, Thomas D. Brock in Biotechnology: A Textbookof Industrial Microbiology, Second Edition (1989) Sinauer Associates,Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem.Biotechnol. 36, 227 (1992). A mutant or variant strain thereof may alsobe identified as having a genome or significant part thereof thathybridizes under conditions of high stringency to the genome of thestrain described herein. The person of skill will be able to recognizehow significant a part of a genome must be so as to consider whether agiven strain is a “mutant or variant thereof.” As such, the person ofskill will reasonably expect that the present invention may be practicedusing such variant or mutant thereof, without undue effort.

As used herein, the expression “effective amount” relates to an amountthat will elicit the desired biological response of a tissue, system oranimal. Such an effective amount of the herein described live F18 E.coli strain can be, for example, but without being limited thereto, theamount that is sufficient for preventing intestinal bacterial infection,minimizing bacterial excretion, preventing a disease caused byintestinal bacterial infection, and the like. The effective amount to beused may vary according to a number of factors. For instance, the numberof factors may be selected from the type of animal, initial weight ofthe animal, growth phase of the animal, environment, feed acceptablecarrier associated with the live strain, i.e., animal facilities, typeand management of production, hygienic status of the facilities, theanimal's stress after weaning or hatching, feed and supplements used,health of the animal and concomitant diseases or treatment, and thelike. For example, the effective amount can be, but without beinglimited thereto, any amount selected within the range of about 5×10⁶ toabout 3×10¹⁰ CFU. The person skilled in the art will be able todetermine a suitable effective amount without undue effort.

As used herein, the term “animal” refers to any young or adult animalsuitable to be used in accordance with the present invention. In onenon-limiting embodiment, the term “animal” refers to a swine. In onenon-limiting embodiment, the term “animal” refers to a pig. In onenon-limiting embodiment, the pig is a pre-weaned pig. In anothernon-limiting embodiment, the pig is a post-weaned pig. The personskilled in the art will be able to determine a suitable animal withoutundue effort.

As used herein, the term “intestinal delivery” refers to a mode ofadministration that enables the strain to eventually reach thegastrointestinal tract, and more preferably the intestines. In onenon-limiting embodiment, the intestinal delivery is performed by oraladministration of the strain. In another non-limiting embodiment, theintestinal delivery is performed by rectal administration, for examplevia a suppository. The person skilled in the art will be able todetermine a suitable mode of administration without undue effort.

As used herein, the expression “feed acceptable carrier” refers to anycarrier, diluent or excipient that is compatible with the hereindescribed strain and can be given to an animal without adverse effects.Suitable feed acceptable carriers known in the art include, but are notlimited to, water, saline, glucose, dextrose, buffered solutions, andthe like. Such a carrier is advantageously non-toxic to the strain andnot harmful to the animal. It may also be biodegradable. The carrier maybe a solid or liquid feed acceptable carrier. A suitable solid feedacceptable carrier is a non-toxic ingestable carrier. For instance, thissolid feed acceptable carrier may be a common solid feedstuff such asthe component of a typical animal diet consisting of cereal products,such as barley meal, maize meal or wheat feed, nut and seed products,such as decorticated ground nut cake or cotton seed cake, or extractedcotton seed cake, together with minor amounts of, for example, feathermeal, seaweed meal, bone meal, bone flour, chalk, salt, urea andvitamins; or it may be an inert solid diluent or carrier of nonutritional value, for example kaolin, talc, calcium carbonate, fuller'searth, attapulgus clay, ground oyster shells or ground limestone; or itmay be starch or lactose. In another specific implementation, the solidfeed acceptable carrier may be ground corn, soybean meal, whey, animalfat, and the like. A suitable liquid feed acceptable carrier is, forexample, water and preferably drinking water; milk such as whole or skimmilk; or a culture medium such as a trypsone soy broth (TSB). The personskilled in the art will be able to determine a suitable feed acceptablecarrier without undue effort.

The scope of the claims should not be limited by the preferredembodiments set forth in the following examples, but should be given thebroadest interpretation consistent with the description as a whole.

EXAMPLES Example 1

a) Non-pathogenic F18 E. coli Strain

The E. coli strain deposited at the International Depositary Authorityof Canada (IDAC) on Jun. 20, 2013 and attributed accession number200613-01 was isolated from feces of a pig in 1996 at the OIE referencelaboratory for Escherichia coli (EcL) at the Faculté de medicinevétérinaire (FMV), Université de Montréal (UdeM), Saint-Hyacinthe,Quebec, Canada by Dr. J. M. Fairbrother.

An identification of the strain was done using the API system. The API20E code for Master Seed is 5004552. Serotyping of the strain revealed aserotype of O141:K94:H—.

Virotyping of the strain was done by colony hybridization and/orpolymerase chain reaction (PCR). Virotyping results showed that thestrain was positive for F18 and AIDA whereas it was negative for thefollowing toxins: STa, STb, LT, EAST1, Stx1, Stx2, CNF, F4, F5, F6, F17,F41, P, AFA, Eae, Paa, Tsh, Aerobactin genes. This strain is thus anon-pathogenic E. coli strain.

The strain is resistant to the following antimicrobials: amoxicillin,ampicillin, clindamycin, doxycycline, erythromycin, neomycin,penicillin, spectinomycin, streptomycin, sulfachlorpyridazin,sulfadimethoxin, sulfathiazole, tiamulin, tilmicosin, tetracycline(chlor and oxy) and tylosin, while being sensitive to the followingantimicrobials: apramycin, ceftiofur, colistine, danofloxacin,enrofloxacin, and gentamicin.

b) Non-pathogenic F4 E. coli Strain

A non-pathogenic F4 E. coli strain may also be used in combination withthe non-pathogenic F18 strain herein described. In one non-limitingembodiment, the non-pathogenic F4 E. coli strain is any non-pathogenicstrain that expresses the F4 determinant. In one non-limitingembodiment, the non-pathogenic F4 E. coli strain is a recombinantstrain, for example the pMK005 strain (Kehoe et al., J Bacteriol. 1983September; 155(3):1071-7). In another non-limiting embodiment, thenon-pathogenic F4 E. coli strain is a natural strain, for example thestrain deposited at the International Depositary Authority of Canada onJan. 21, 2005 and attributed accession number IDAC 210105-01, asdescribed in U.S. Pat. No. 7,981,411 (the entire contents thereof areherein incorporated by reference in their entirety).

Example 2

In a broad aspect, the purpose of this study was to evaluate the impactof different oral doses of the herein described non-pathogenic F18strain on fecal excretion thereof and on anti-F18 systemic humoralresponses.

The following Table 1 summarizes generally the study schedule:

Day Event 0 Arrival of pigs in the experimental unit and distributioninto 3 groups 0-5 Acclimatization period 2 and 5 to 19 Fecal samplingfor PCR 5 Vaccination 5 to 8, 10, 12, Fecal sampling for viable count ofvaccine 14, 16, 18 and 19 strain (based on hemolytic colonies) 5, 10,12, 15 and 19 Blood sample 8 First necropsy 19  Final necropsy and endof experimentI—Study Designa) Animals

Blood samples of 50 nursing piglets were taken for F18 receptor statusdetection analysis and piglets were ear tagged. Selected weaned pigs(males and females), aged between 17 and 20 days, were purchased from alocal farm located in the Montérégie, Québec, Canada. Animals weretransported to the isolation facilities. The farm selected showed norecent (less than 6 months) Porcine Reproductive and RespiratorySyndrome (PRRS) and post-weaning disease episodes. The pigs were healthypigs and weighted between 4 to 6 Kg. A total of twenty one (21) pigs,positive for F18-receptor status, were used, randomly separated usingear tag numbers in three (3) groups of 7 animals (three different F18composition doses). At day 2 post-weaning, a fecal sample was taken todetermine the existing colonisation with F4 positive and F18 positivestrains using PCR. No animal was found to be positive for either F4positive or F18 positive bacteria at day 2 post-weaning.

b) Housing and Feeding

Treatment groups were held in separate rooms. Animals had ad libitumaccess to water and were fed twice daily during the study. The animalsdiet was constituted of a high soybean meal as described in U.S. Pat.No. 6,355,859 (the entire content thereof is herein incorporated byreference in its entirety). No concurrent medication or vaccination wasadministered during the study.

c) Strain Preparation and Administration

Two growth curves were prepared the same way to evaluate the timerequired by the non-pathogenic F18 strain to attain its optimal yield.Working Seed (WS) was used to prepare the growth curve and the culturethat was used for preparing the F18 composition. One milliliter of theWS was used to inoculate 500 mL of TrypticSoy Broth without materials ofanimal origin. The culture was incubated approximately 6.5 hours at 37°C. with agitation (180 rpm). A viable count was done to evaluate theyield of the F18 strain culture and a PCR reaction was used todemonstrate that the appropriate strain was used during the assay. Basedon the results, the culture was adjusted to meet the doses to be testedusing sterile TSB without materials of animal origin: Group 1, 5×10⁸CFU/dose; Group 2, 1×10⁹ CFU/dose; and Group 3, 5×10⁹ CFU/dose.

Nine (9) doses of non-pathogenic F18 strain composition were preparedfor each formulation, 2 more than the number of piglets to be treated tocompensate for any composition loss. Six (6) mL of F18 composition wereadministered orally to every piglet. The F18-composition wasadministered at day 5 post-weaning using an esophageal tube.

II—Observations and Evaluation

a) General Health

Piglets were observed twice daily for general health: behavior,dehydration, appetite, and general physical condition. Mobility and hairaspect were also evaluated.

b) Diarrhea

Fecal consistency are scored daily as follow: 0, normal; 1, pasty; 2,presence of liquid but more solid particles than liquid; 3, presence ofmore liquid than solid particles; and 4, totally liquid.

c) Excretion of the Administered Non-pathogenic F18 Strain

At day 2 and days 5 to 19 post-weaning, feces were tested by PCR(multiplex: LT, STa, STb and F4; F18, Stx2e and AIDA) followingenrichment in Luria-Bertoni (LB) broth to evaluate the presence of theadministered non-pathogenic F18 strain in each group. A sample positivefor F18 was considered positive for the administered non-pathogenic F18strain. At days 5 to 8, 10, 12, 14, 16, 18 and 19 post-weaning, a viablecount was done on feces samples using Tryptic Soy Agar II (TSA II)—5%sheep blood to evaluate the level of the administered non-pathogenic F18strain excreted after administration. Only haemolytic colonies withtypical E. coli morphology were counted.

d) Immunization Evaluation

Blood samples were collected at days 5, 10, 12, 15 and 19 post-weaningto evaluate by ELISA the optimal immune response accordingly to thedosage of the administered non-pathogenic F18 strain.

III—Necropsy

a) Necropsy

A first necropsy was conducted 3 days post-vaccination, at day 8post-weaning on 2 animals chosen randomly, and gross abnormalities areevaluated. The second necropsy was conducted at the end of the study,thus on day 19 post-weaning, which was 14 days post-vaccination on allthe remaining animals.

b) Intestinal Content Consistency

Intestinal content consistency are scored in the jejunum, ileum, caecum,colon and rectum as follow: 0, normal; 1, pasty; 2, presence of liquidbut more solid particles than liquid; 3, presence of more liquid thansolid particles or totally liquid.

c) Colonization of the Ileum by the Vaccine Strain

A 2 centimeters portion of the ileum was sampled at 10 cm proximal ofthe ileocaecal junction. Live bacterial enumeration was performed usingTSA II—5% sheep blood, where only haemolytic colonies with typical E.coli morphology were counted.

IV—Results

No mortality was observed in both treated and control animals throughoutthe study. All pigs selected were positive for F18 receptor as per thePCR-RFLP method. In general, piglets were in good health all along thestudy. Few animals showed reduced mobility that appeared beforeadministration of the non-pathogenic F18 strain or due to injuries thusnot related to the administration of the non-pathogenic F18.

During this study, a score of 2 or more was considered as diarrhea. Onthe 21 animals, 5 showed transient mild diarrhea after F18administration. One animal (pig 5 in group 3) had diarrhea (score of 3)for 2 consecutive days (Tables 2a, 2b and 2c).

Fecal samples were tested daily by PCR to detect presence of the F18administered strain; 3 animals in group 2 (1×10⁹ CFU/dose) were positivefor the F18 administered strain, at the latest, on day 11post-vaccination, where group 3 (5×10⁹ CFU/dose) had 1 piglet positiveat day 10, 1 at day 8 and 3 at day 7 post-vaccination. For group 1(5×10⁸ CFU/dose) 1 piglet was positive for the non-pathogenic F18administered strain at day 8 post-vaccination, 1 piglet at day 7 and 2at day 6 post-vaccination (Tables 2a, 2b and 2c).

TABLE 2a Scores, duration and severity of diarrhea for groupadministered with 5 × 10⁸ CFU/dose

TABLE 2b Scores, duration and severity of diarrhea for groupadministered with 1 × 10⁹ CFU/dose

TABLE 2c Scores, duration and severity of diarrhea for groupadministered with 5 × 10⁹ CFU/dose

^(*1)Fecal consistency are scored as follows: 0, normal; 1, pasty; 2,presence of liquid but more solid particles than liquid; 3, presence ofmore liquid than solid particles; and 4, totally liquid; ^(*2)Day 5: F18administered strain administration; ^(*3)Duration of diarrhea (Number ofdays with scores of 2 or more for the post-vaccination period; days 6 to19); ^(*4)Severity of diarrhea (Cumulative diarrhea scores for the postvaccination period; days 6 to 19); ^(*5)Median of diarrhea score. Thevertical bold line designates the last day that F18 (the vaccine strain)was detected by PCR. ND: Not done E: empty D: Deceased (subject tonecropsy)

-   The vertical bold line designates the last day that F18 (the vaccine    strain) was detected by PCR. ^(*1) Fecal consistency are scored as    follows: 0, normal; 1, pasty; 2, presence of liquid but more solid    particles than liquid; 3, presence of more liquid than solid    particles; and 4, totally liquid;^(*2) Day 5: F18 administered    strain administration;^(*3) Duration of diarrhea (Number of days    with scores of 2 or more for the post-vaccination period; days 6 to    19);^(*4) Severity of diarrhea (Cumulative diarrhea scores for the    post-vaccination period; days 6 to 19);^(*5) Median of diarrhea    score.-   ND: Not done-   E: empty-   D: Deceased (subjected to necropsy)

Fecal excretion of the non-pathogenic F18 administered strain was alsoevaluated by bacterial enumeration from fecal samples at day 5 to 8, 10,12, 14, 16, 18 and 19. FIG. 1 shows the excretion of the strainpost-administration. The level of bacteria excreted with the lower dose(5×10⁸ CFU) is inferior to the two other, the highest excretion beingobserved with animals from group 2, administered with a dose of 1×10⁹CFU.

No gross abnormalities were observed for internal organs. Intestinalcontents consistencies were evaluated for accumulation of fluid.

In this study, a score of 2 or more was considered as fluid accumulationin an intestinal segment at day of necropsy. Tables 3a, 3b and 3c showthat one animal in group 1 presented two consecutive segments with fluidaccumulation (score of 2). The other animals did not show fluidaccumulation.

No undesirable event related to administration of the non-pathogenic F18strain was observed during the study, indicating that the tested dosageswere generally safe.

Based on the strain excretion evaluation, both PCR and bacteriaenumeration suggested that the 1×10⁹ CFU/dose is a suitable dosage forobtaining prolonged fecal excretion of this strain. Both testsdemonstrated that administration of the non-pathogenic F18 strain at1×10⁹ CFU/dose was excreted on a longer period than the other testeddosages.

TABLE 3a Intestinal content consistency scores and extent and severityof accumulation of fluid in the intestines for group administered with 5× 10⁸ CFU/dose Necropsy Intestinal content fluid score^(*1) Extent^(*2)Severity^(*3) ID day Jejunum Ileon Caecum Colon Rectum (Max = 5) (Max =15) 9 8 0 E 1 2 2 2 5 18 19 0 0 0 0 0 0 0 23 19 0 0 0 0 0 0 0 27 19 0 00 0 0 0 0 30 8 0 E 0 0 0 0 0 33 19 0 0 0 0 0 0 0 50 19 0 0 0 0 0 0 0MD^(*4) day 8 0 E 0.5 1 1 1.5 3.5 MD^(*4) day 19 0 0 0 0 0 0 0

TABLE 3b Intestinal content consistency scores and extent and severityof accumulation of fluid in the intestines for group administered with 1× 10⁹ CFU/dose Necropsy Intestinal content fluid score^(*1) Extent^(*2)Severity^(*3) ID day Jejunum Ileon Caecum Colon Rectum (Max = 5) (Max =15) 6 19 0 0 0 1 0 0 1 26 19 0 0 0 0 0 0 0 29 19 0 0 0 1 0 0 1 34 8 0 00 0 0 0 0 37 8 0 0 0 0 0 0 0 42 19 0 E 0 0 0 0 0 45 19 0 E 0 0 0 0 0MD^(*4) day 8 0 0 0 0 0 0 0 MD^(*4) day 19 0 0 0 0 0 0 0

TABLE 3c Intestinal content consistency scores and extent and severityof accumulation of fluid in the intestines for group administered with 5×10⁹ CFU/dose Necropsy Intestinal content fluid score^(*1) Extent^(*2)Severity^(*3) ID day Jejunum Ileon Caecum Colon Rectum (Max = 5) (Max =15) 4 19 1 0 0 0 0 0 1 5 19 0 1 0 1 1 0 3 13 19 0 E 1 1 0 0 2 17 8 0 0 00 0 0 0 21 19 1 E 1 0 0 0 2 46 8 0 0 0 0 0 0 0 49 19 0 0 0 0 0 0 0MD^(*4) day 8 0 0 0 0 0 0 0 MD^(*4) day 19 0 0 0 0 0 0 2 ^(*1)Intestinal content consistency are scored in the jejunum, ileum, caecum,colon, and rectum, as follows: 0, normal; 1, pasty; 2, presence ofliquid but more solid particles than liquid; 3, presence of more liquidthan solid particles or totally liquid. ^(*2) Extent of accumulation offluid in the intestines (Number of intestinal segments with scores of 2or more) ^(*3) Severity of diarrhea (Cumulative consistency intestinalcontent scores for the intestinal segments) ^(*4) Median of intestinalcontent consistency score ND: Not done

Example 3

In a broad aspect, the purpose of this study was to evaluate theefficacy of the combined composition, including the herein describednon-pathogenic F4 and F18 E. coli strains.

The following Table 4 summarizes generally the study schedule:

DAY EVENT 0 Arrival of pigs in the experimental unit and distributioninto 2 groups 0 to 5 Acclimatization period 1, 12, 14, 16, 18 and 19Fecal sampling for PCR 4, 12, 17 and 19 Blood sample 5 Oraladministration of dose or placebo 12, 13 and 14 Challenge 12, 14, 16, 18and 19 Fecal sampling for viable count of challenge strain 19  Finalnecropsy and end of experimentI—Study Designa) Animals

Blood samples of 50 nursing piglets were taken for F18 receptordetection analysis and piglets were ear tagged. Selected weaned pigs(males and females), aged between 17 and 21 days, were purchased from acommercial farm located in the Montérégie area, Québec, Canada. Animalswere transported to the isolation facilities. The farm showed no recent(less than 6 months) Porcine Reproductive and Respiratory Syndrome(PRRS) and post-weaning disease episodes. The pigs were healthy pigs andweighted between 4 to 6 Kg.

A total of twenty (20) pigs were used, randomly separated using ear tagnumbers. A randomizer software was used to randomly distribute the 20animals in two (2) groups of 10 animals (Control and administeredgroup). At day 1 post-weaning, a fecal sample was taken to determine theexisting colonization with pathogenic F4-ETEC positive strain andF18-ETEC/STEC positive strains originating from the farm using PCRanalysis. Genes targeted are STa, STb, LT, F4, Stx2 and F18. No animalpositive for either F4-ETEC or F18-ETEC/STEC bacteria at day 1post-weaning was found.

b) Housing and Feeding

Treated and control groups were held in separate rooms. Animals had adlibitum access to water and feed during the study. The animals diet wasconstituted of a high soybean meal as described in U.S. Pat. No.6,355,859. No concurrent medication or vaccination was administeredduring the study.

c) Strain Preparation and Administration

The rehydrated combined composition was diluted to obtain a minimum of5×10⁸ CFU/strain per 6-mL dose. The placebo was constituted of 6 mL ofsterile water. The combined composition and placebo were orallyadministered using a syringe with rubber tubing.

d) Challenge Strain (F18-STEC EcL14724) Preparation and Administration

This challenge strain was deposited at the International DepositaryAuthority of Canada (IDAC) on Jul. 9, 2013 and attributed accessionnumber 090713-01.

The culture was prepared to obtain an infectious dose of 1×10¹¹ CFU perpiglet. Following the administration of 10 mL of calcium carbonate 1.2%(CaCO₃), five (5) mL of the challenge strain were orally administered toevery piglet using an esophageal tube. The challenge strain wasadministered during three (3) consecutive days, i.e. day 12, 13 and 14post-weaning at a concentration of 1×10¹¹ CFU/dose every day.

e) Observations and Evaluation

Piglets were observed twice daily for general health: behaviour,appetite, general physical condition, mobility and hair aspect wasevaluated. Dehydration and body temperature was observed once a day.Excretion of the challenge strain was evaluated by PCR and viablebacterial count (CFU). Blood samples were analyzed using specificEnzyme-Linked Immunosorbent assays (ELISA). Results were presented aspercentage of positivity (PP %) versus the positive control.

II—Necropsy

Colonization of the ileum and the caecum by the challenge strain wasdetermined as follows. A 2 centimeters portion of the ileum was sampledat 10 cm proximal of the ileocaecal junction as well as a portion of thecaecum. Live bacterial enumeration was performed using TSA II—5% sheepblood, where only haemolytic colonies with typical E. coli morphologywere counted after 12 to 18 hours incubation at 37° C.

III—Results

a) General Information

From the 50 piglets tested, 30 were susceptible to F18 strain adhesionand 20 animals were randomly selected for the trials. In general,piglets were in good health before the challenge.

The following Table 5 shows PCR results of excreted challenge strainfrom feces of treated and control animals:

Pigs (%) excreting the challenge strain at day post-last challenge (daypost-weaning) 0 (14) 2 (16) 4 (18) 5 (19) Placebo 100% 100% 67% 56%Combined 100%  60%  0%  0% composition

As demonstrated in Table 5, administration of the combined compositionreduced excretion of the challenge strains compared to pigs whichreceived a placebo. More than half of the placebo animals (56%) werestill excreting the challenge strain 5 days after the last challenge(end of study) while the challenge strain was not detected in animalstreated with the combined composition as early as 4 days post-lastchallenge.

FIG. 2 shows viable bacterial count (CFU)/g of excreted challenge strainfrom feces of treated and control animals. A peak in the concentrationof the excreted challenge strain is seen at post-weaning day 14 (0 daypost-challenge) for both treated and control animals. In treatedanimals, the concentration of the challenge strain in feces started todecrease at day 2 post-last challenge (post-weaning day 16), and furtherreductions were observed compared to placebos at days 4 and 5 post-lastchallenge. These results are in accordance with the PCR results. Moreprecisely, administration of the combined composition reduced theexcretion level of the challenge strain by about 2 log at 5 dayspost-last challenge (end of study).

b) Ileum and Caecum Colonization by the Challenge Strain

Differences for intestinal colonization between the treated and placeboanimals were observed for both intestinal tissue segments (ileum andcaecum). FIG. 3 demonstrates that administration of the combinedcomposition significantly reduced the colonization of the intestine bythe challenge strain. More precisely, administration of the combinedcomposition reduced the challenge strain colonization of the ileum andcaecum by about 2 log at 5 days post-last challenge (end of study).

c) Immune Response Evaluation

Presence of systemic IgM against F18 fimbriae was evaluated in serumsamples using specific ELISA. The combined composition treated animalsshowed a specific immune response as soon as 7 days post administration(FIG. 4).

Based on the resulting reduction of the challenge strain colonization,reduction of excretion and stimulation of specific anti-F18 antibodies,this study suggests that intestinal delivery of a composition includingthe herein described non-pathogenic F18 strain is capable of preventingF18 E. coli intestinal bacterial infection.

Example 4

In a broad aspect, the purpose of this study was to evaluate theefficacy of the herein described non-pathogenic F18 E. coli strain.

I—Study Design

a) Animals

Prior to the entry of animals into the facilities, ten nursing pigletsof 10 litters, for a total of 100 piglets, were sampled at the farrowingunit for DNA isolation and screened for F18 receptor (RF18+) status by aRFLP-PCR method. Thirty (30) RF18+ piglets were randomly distributedbetween groups by blocking by litter (1:1 ratio), with generation ofblock size of n=6. Piglets were allocated to one of the followinggroups: T1 which were administered the non-pathogenic F18 tested strain(n=15); and T2 which were administered a placebo (n=15).

Animals of the treated group (T1) were comingled into a pen of anexperimental room and animals of the placebo group (T2) were comingledinto a pen of another experimental room until the challenge phase. Onthe day of the challenge, that is 11 days after administration of thenon-pathogenic F18 strain or placebo, animals of T1 (n=15) and T2 (n=15)groups were transferred, prior to challenge, into 2 separate penslocated in a challenge room. All 15 pigs of a particular treatment groupwere commingled in the respective pen. Throughout the study, animalshave ad libitum access to water, except during withholding of the waterprior to vaccination. Animals are fed twice daily with non-medicatedhigh soybean meal (U.S. Pat. No. 6,355,859).

At day 1 post-weaning, a fecal sample was taken to determine theexisting colonization with pathogenic F4-ETEC positive strain andF18-ETEC/STEC positive strains originating from the farm using PCRanalysis. Genes targeted are STa, STb, LT, F4, Stx2 and F18. No animalpositive for either F4-ETEC or F18-ETEC/STEC bacteria at day 1post-weaning was found. Three (3) animals of the T1 group were withdrawnfrom the study for animal welfare reasons. These adverse events wereconsidered not being related to administration of the non-pathogenic F18strain.

The following Table 6 summarizes generally the animals used:

Species: Domestic pigs (Pen Ar Lan Naïma). This swine breed is a hybridbreed commercialized in America, Europe and Asia. Number and Sex: 30animals; Castrated 30% male and 70% female. Supplier: Commercial farm,Montérégie, Québec, Canada Age when tested: The test article wasintended for administration of test strain of piglets from approximately17 days of age. Weight range at study 3.120-7.295 Kg initiation:Acclimatisation: 1 day Medication: No concurrent medication orvaccination was administered within 3 days prior to and during the studyto any of the study animals

The following Table 7 generally summarizes the study design:

Day of activity T1 T2 Day −2 Arrival of RF18+ pigs in the isolationfacilities and random allocation of pigs, general health control andweight Day 0 (approx. non-pathogenic F18 Placebo 17 days of age)administration Days 1-11 Daily health control Day 11-12-13 Challengewith F8-STEC Day 11 to 18 Observations for health Day 18 Necropsy andmicrobiologyII—Procedure and Evaluation

T1 group and T2 group animals could not be commingled due to the natureof the tested product (i.e., a live vaccine) requiring treated animalsto be housed separately from placebo controls. The non-pathogenic F18strain and placebo solutions were prepared by persons not involved inthe animal phase. A code “A” and “B” was randomly assigned to thenon-pathogenic F18 strain and placebo solutions. Rooms were randomlyallocated to treatment and identified as rooms 1 and 2. Each room hadall T1 group or all T2 group animals commingled in a single pen. Thepersonnel involved in the animal phase and doing laboratory analysiswere blinded to treatment and room allocations. For the necropsy phase,the sequence of animals to be necropsied was randomly assigned andanimals were re-identified as A to AD. The person performing thenecropsy was blinded to pig identification and treatment. Therandomization codes were kept in a sealed envelope by thebiostatistician in charge.

a) Preparation of the Non-pathogenic F18 Strain and Administration

The non-pathogenic F18 strain was cultured in a fermenter, then thebacteria was frozen and stored at −78° C. in 10 mL glass vialscontaining 200 nominal doses. The frozen bacteria were diluted withwater at room temperature prior to use.

The dose was administered once at one day post-weaning (Day 0) atapproximately 3×10⁸ CFU per 2-mL dose. The dose was administered orallyusing a syringe mounted with a rubber tube. The control animals receivedthe same amount of placebo solution (water).

b) Challenge Strain (F18-STEC Strain EcL 14724)

The challenge strain used in this assay was re-isolated from a piglet(#381) previously challenged with the EcL 14724 strain (positive forStx2, F18ab, AIDA and Paa). The challenge strain was orally administeredonce at the 3 consecutive days 11, 12 and 13 of the study atapproximately 1×10¹⁰ CFU per pig. Each challenge dose was administeredusing a gelatin capsule as a replacement for oesophagic intubation.Carbadox (an antibiotic stimulating the release of the shigatoxin) wasorally administered in a gelatin capsule immediately afteradministration of the challenge strain to trigger the release of theShigatoxin.

c) General Health Evaluation

Pigs were observed once daily prior to the challenge period for generalhealth. Following the first challenge, animals were monitored twicedaily for general health, including depression, dehydration, appetiteand for any clinical signs associated to edema disease: ataxia, facialor limb edema, sudden death.

d) Evaluation of the Shedding of the Challenge Strain

Fecal shedding of the challenge strain was quantified using aMost-Probable-Number PCR (MPN-PCR) method targeting the gene coding forthe FedA major protein of the F18 fimbriae.

e) Necropsy

Necropsies were carried out at 7 days post-first challenge. Colonizationof the caecum by the challenge strain was quantified using aMost-Probable-Number PCR method targeting the gene coding for the FedAmajor protein of the F18 fimbriae using approximately 2 cm² portion ofthe tissue.

III—Results

PCR analysis demonstrated the efficacy of the administration of thenon-pathogenic F18 strain to prevent excretion of the challenge F18shigatoxigenic pathogenic strain. A reduction of about 2.5 log of fecalshedding was observed for the treated animals compared to the placeboanimals at 4 days post-last challenge (Table 8). At 4 days post lastchallenge, 93% of placebo animals shed more than 1×10⁵ CFU of thechallenge strain per gram of fecal sample while only 42% of treatedanimals did (Table 9).

The following Table 8 shows shedding of the challenge strain at day 4post-last challenge:

Median of shedding (CFU of the challenge strain/g of fecal sample)Placebos animals 7.8 × 10⁶ Treated animals 5.2 × 10⁴

The following Table 9 shows the proportion of pigs shedding thechallenge strain at different bacterial load at day 4 post-lastchallenge:

CFU of the challenge strain/g of fecal samples <1 × 10⁴ 10⁴ 10⁵ >1 × 10⁶Placebo animals  0%  7% 33% 60% Treated animals 25% 33% 17% 25%

A reduction of about 1 log of the challenge strain detected in thecaecum was observed for the treated animals (administered thenon-pathogenic F18 strain) compared to the placebo animals at 5 dayspost-last challenge (Table 10). At 5 days post-last challenge, more than53% of placebo animals shed more than 1×10⁵ CFU of the challenge strainper gram of caecum tissue while only 8% of treated animals did (Table11). More than 80% of placebo animals were colonized in the caecum bymore than 1×10⁴ CFU of the challenge strain per gram of caecum comparedto 50% of treated animals at 5 days post-last challenge (Table 11).

The following Table 10 shows colonization of the caecum by the challengestrain at day 5 post-last challenge:

Median of caecum colonization (CFU of the challenge strain/g of caecumtissue) Placebo animals 3.4 × 10⁵ Treated animals 1.5 × 10⁴

The following Table 11 shows the proportion of pigs of which the caecumwas colonized by the challenge strain at different bacterial load at day5 post-last challenge:

CFU of the challenge strain/g of caecum samples <1 × 10² 10² 10³ 10⁴ >1× 10⁵ Placebo animals 7%  7%  7% 27% 53% Treated animals 8% 17% 25% 42% 8%

Based on the resulting reduction of the challenge strain colonizationand reduction of excretion, this study suggests that intestinal deliveryof a composition including the non-pathogenic F18 strain is capable ofpreventing F18 E. coli edema-causing intestinal bacterial infection.

The following clauses provide a further description of examples ofnon-limiting embodiments in accordance with the present invention:

Clauses

-   Clause 1: Isolated E. coli strain deposited at the International    Depositary Authority of Canada (IDAC) on Jun. 20, 2013 and    attributed accession number 200613-01.-   Clause 2: The isolated E. coli strain of clause 1, in lyophilized    form.-   Clause 3: The isolated E. coli strain of clause 1, in frozen form.-   Clause 4: The isolated E. coli strain of clause 1, wherein the    strain is in association with a feed acceptable carrier.-   Clause 5: A composition comprising the isolated E. coli strain of    any one of clauses 1 to 3 and a feed acceptable carrier.-   Clause 6: The composition of clause 5, further comprising an    isolated live non-pathogenic E. coli that expresses the F4 fimbriae.-   Clause 7: The composition of clause 6, wherein said    non-pathogenic E. coli that expresses the F4 fimbriae is the strain    deposited at the International Depositary Authority of Canada on    Jan. 21, 2005 and attributed accession number IDAC 210105-01.-   Clause 8: A method for preventing F18 pathogenic E. coli intestinal    infection in an animal, comprising intestinal delivery to the animal    of an effective amount of a live E. coli strain deposited at the    International Depositary Authority of Canada (IDAC) on Jun. 20, 2013    and attributed accession number 200613-01-   Clause 9: The method of clause 8, wherein said intestinal delivery    is obtained by oral administration of the live E. coli strain.-   Clause 10: The method of clause 8 or 9, wherein said live E. coli    strain is in lyophilized form.-   Clause 11: The method of clause 8 or 9, wherein said live E. coli    strain is in frozen form.-   Clause 12: The method of clause 8 or 9, wherein said live E. coli    strain is in association with a feed acceptable carrier.-   Clause 13: The method according to any one of clauses 8 to 12,    wherein said effective amount is of at least 5×10⁷ CFU.-   Clause 14: A method according to any one of clauses 8 to 12, wherein    said animal is a pig.-   Clause 15: A method for preventing edema disease or diarrhea caused    by an F18 pathogenic E. coli infection in an animal, comprising    intestinal delivery to the animal of a live E. coli strain deposited    at the International Depositary Authority of Canada (IDAC) on Jun.    20, 2013 and attributed accession number 200613-01.-   Clause 16: The method of clause 15, wherein said intestinal delivery    is obtained by oral administration of the live E. coli strain.-   Clause 17: The method of clause 15 or 16, wherein said live E. coli    strain is in lyophilized form.-   Clause 18: The method of clause 15 or 16, wherein said live E. coli    strain is in frozen form.-   Clause 19: The method of clause 15 or 16, wherein said live E. coli    strain is in association with a feed acceptable carrier.-   Clause 20: The method according to any one of clauses 15 to 19,    wherein said effective amount is of at least 5×10⁷ CFU.-   Clause 21: A method according to any one of clauses 15 to 20,    wherein said animal is a pig.-   Clause 22: Use, for preventing an F18 pathogenic E. coli intestinal    infection in an animal, of an effective amount of an effective    amount of a live E. coli strain deposited at the International    Depositary Authority of Canada (IDAC) on Jun. 20, 2013 and    attributed accession number 200613-01, the strain being adapted for    intestinal delivery in the animal.-   Clause 23: Use, in the manufacture of a composition for use in    preventing an F18 pathogenic E. coli intestinal infection in an    animal, of an effective amount of a live E. coli strain deposited at    the International Depositary Authority of Canada (IDAC) on Jun. 20,    2013 and attributed accession number 200613-01, the composition    being adapted for intestinal delivery in the animal.-   Clause 24: Use, for preventing edema disease or diarrhea caused by    an F18 pathogenic E. coli infection in an animal, of an effective    amount of an effective amount of a live E. coli strain deposited at    the International Depositary Authority of Canada (IDAC) on Jun. 20,    2013 and attributed accession number 200613-01, the strain being    adapted for intestinal delivery in an animal.-   Clause 25: Use, in the manufacture of a composition for use in    preventing edema disease or diarrhea caused by an F18 pathogenic E.    coli infection in an animal, of an effective amount of an effective    amount of a live E. coli strain deposited at the International    Depositary Authority of Canada (IDAC) on Jun. 20, 2013 and    attributed accession number 200613-01, the composition being adapted    for intestinal delivery in an animal.-   Clause 26: The use of any one of clauses 22 to 25, wherein said    live E. coli strain is adapted for oral administration.-   Clause 27: The use of any one of clauses 22 to 26, wherein said    live E. coli strain is in lyophilized form.-   Clause 28: The use of any one of clauses 22 to 26, wherein said    live E. coli strain is in frozen form.-   Clause 29: The use of any one of clauses 22 to 26, wherein said    live E. coli strain is in association with a feed acceptable    carrier.-   Clause 30: The use according to any one of clauses 22 to 29, wherein    said effective amount is of at least about 5×10⁷ CFU.-   Clause 31: The use according to any one of clauses 22 to 30, wherein    said animal is a pig.-   Clause 32: The isolated E. coli strain of any one of clauses 1 to 4,    for use in preventing an F18 pathogenic E. coli intestinal infection    in an animal, the strain being adapted for intestinal delivery in    the animal.-   Clause 33: The isolated E. coli strain of any one of clauses 1 to 4,    for use in the manufacture of a composition for use in preventing an    F18 pathogenic E. coli intestinal infection in an animal, the    composition being adapted for intestinal delivery in the animal.-   Clause 34: The isolated E. coli strain of any one of clauses 1 to 4,    for use in preventing edema disease or diarrhea caused by an F18    pathogenic E. coli infection in an animal, the strain being adapted    for intestinal delivery in the animal.-   Clause 35: The isolated E. coli strain of any one of clauses 1 to 4,    for use in the manufacture of a composition for use in preventing    edema disease or diarrhea caused by an F18 pathogenic E. coli    infection in an animal, the composition being adapted for intestinal    delivery in the animal.

Note that titles or subtitles may be throughout the present forconvenience of a reader, which in no way should limit the scope of theinvention. Moreover, certain theories are proposed and disclosed herein;however, in no way they, whether they are right or wrong, should limitthe scope of the invention so long as the invention is practicedaccording to the present disclosure without regard for any particulartheory or scheme of action.

It will be understood by those of skill in the art that throughout thepresent specification, the term “a” used before a term encompassesembodiments containing one or more to what the term refers. It will alsobe understood by those of skill in the art that throughout the presentspecification, the term “comprising”, which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, un-recited elements ormethod steps.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions will control.

While the herein described studies have used weaned pigs, the person ofskill will readily understand that the pigs being treated mayalternatively be pre-weaning pigs.

As used herein, “around”, “about” or “approximately” shall generallymean within 20 percent, preferably within 10 percent, and morepreferably within 5 percent of a given value or range. Numericalquantities given herein are generally approximate; meaning that the term“around”, “about” or “approximately” can be inferred if not expresslystated.

All references cited throughout the specification are herebyincorporated by reference in their entirety.

The scope of the claims should not be limited by the preferredembodiments set forth in the examples, but should be given the broadestinterpretation consistent with the description as a whole.

The invention claimed is:
 1. A composition for animal consumption,comprising a live Escherichia coli strain deposited at the InternationalDepositary Authority of Canada (IDAC) on Jun. 20, 2013, and attributedaccession number 200613-01 and a feed acceptable carrier, thecomposition being in lyophilized form and the strain being in an amountsufficient for administration of from about 5×10⁶ to about 3×10¹⁰ CFUper animal and for protecting the animal from disease caused by F18infection.
 2. The composition for animal consumption according to claim1, the strain being in an amount sufficient for administration of 5×10⁸CFU per animal.
 3. The composition for animal consumption according toclaim 1, the strain being in an amount sufficient for administration of1×10⁹ CFU per animal.
 4. The composition for animal consumptionaccording to claim 1, the strain being in an amount sufficient foradministration of 5×10⁹ CFU per animal.
 5. The composition for animalconsumption according to claim 1, the strain being in an amountsufficient for administration of 3×10⁸ CFU per animal.
 6. Thecomposition for animal consumption according to claim 1, wherein theanimal is a pig.
 7. The composition for animal consumption according toclaim 6, wherein the pig is a post-weaning pig.
 8. A composition forintestinal delivery in an animal, comprising a live Escherichia colistrain deposited at the International Depositary Authority of Canada(IDAC) on Jun. 20, 2013, and attributed accession number 200613-01 and afeed acceptable carrier, the composition being in lyophilized form andthe strain being in an amount sufficient for administration of fromabout 5×10⁶ to about 3×10¹⁰ CFU per animal and for protecting the animalfrom disease caused by F18 infection.
 9. The composition according toclaim 8, the strain being in an amount sufficient for administration of5×10⁸ CFU per animal.
 10. The composition according to claim 8, thestrain being in an amount sufficient for administration of 1×10⁹ CFU peranimal.
 11. The composition according to claim 8, the strain being in anamount sufficient for administration of 5×10⁹ CFU per animal.
 12. Thecomposition according to claim 8, the strain being in an amountsufficient for administration of 3×10⁸ CFU per animal.
 13. Thecomposition according to claim 8, wherein the animal is a pig.
 14. Thecomposition according to claim 13, wherein the pig is a post-weaningpig.
 15. The composition according to claim 8, wherein the compositionis adapted for reconstitution in water.